Abstract This proposal focuses on the rational design of antigens for serodiagnosis of Zika virus (ZIKV) with minimal cross-reactivity to other human pathogenic flaviviruses. Zika virus (ZIKV) infection can cause miscarriage, microcephaly, and Guillain-Barr syndrome. A specific serodiagnosis of ZIKV infection is urgently needed to track the spread of ZIKV infection and to diagnose individuals exposed to ZIKV in scenarios such as prenatal diagnosis and neonatal monitoring, blood donor screening, screening of travelers returning from Zika-endemic regions. ZIKV serodiagnosis is highly challenging because of extensive cross-reactivity with dengue and other flaviviruses that co-circulate with ZIKV. Our previous and ongoing work defining the human serotype specific antibody responses to dengue envelope (E) protein place us in a unique position to define similar regions in ZIKV E and produce candidate diagnostic antigens for serodiagnostics. Fortified with detailed structural information, we will rationally design and produce substructures of Zika E protein bearing unique antigenic sites for specific and sensitive serological detection of ZIKV infections in flavivirus nave, flavivirus pre-immune or flavivirus-vaccinated populations. For aim 1, we will produce ZIKV candidate antigens and measure their antibody binding response across a reference panel of immune sera from people exposed to primary or secondary flavivirus infections, including ZIKV and DENV. In Aim 2, we will use protein engineering approaches to redesign most efficacious candidate antigens to change cross-reactive antigenic sites, while preserving Zika specific sites, to improve the diagnostic potential of antigens for differential serodiagnosis of Zika infection. We will compare and contrast one or a combination of surface redesigned Zika antigens for serological detection of recent and prior exposure to ZIKV. In follow up studies, we will partner with reference laboratories and commercial partners to use recombinant protein antigens to develop simple assays for use at the point of care and in surveillance programs.